The conservation of the molecular and embryological processes orchestrating development in vertebrate and invertebrate species is often exploited to identify conserved gene function in other species, yet orthologous data sets are frequently neglected when building biological networks and cis-regulatory models that predict gene expression patterns. The latter omission is surprising since the convergence of relevant data sets from orthologous species should produce a stronger model with improved predictive power. Recent studies have characterized the histone mark distribution and expression profiles of differentiating human and mouse ESCs to uncover the cis-regulatory elements (CREs) and transcript patterns that characterize the mammalian cardiac lineage. Here we used these datasets to generate a predictive epigenetic and gene expression signature of candidate genes that control mouse and human CM differentiation from ESCs. We reasoned that the histone modifications and gene expression patterns associated with genes critical for CM differentiation will show marks of repression during the ESC state and marks of activation when pluripotent cells become restricted as cardiac precursors (CPs). Using this epigenetic and gene expression signature, we show that the overlapping mouse and human genes are the best candidates for having cardiac regulatory activities because this group contains genes showing greater evolutionary conservation and enrichment for cardiogenic function and relevant gene ontology functional categories. To evaluate the potential cardiogenic activities of the identified mammalian genes, we initially undertook a large-scale, whole embryo RNAi-based screen of the corresponding Drosophila orthologs. We reasoned that a primary screen in Drosophila would increase the efficiency of the screen as key regulators are more likely to be evolutionarily-conserved and significantly diminish the cost associated with functional testing in differentiating human ESCs. This approach uncovered dozens of previously unrecognized regulators of fly heart development. Targeted knockdown of two of these genes in differentiating ESCs revealed their necessity for the formation of functional CMs. Current work using ChIP-seq and RNA-seq techniques is directed toward investigating the transcriptional networks acting downstream of these two TFs, as well as for assessing the functions of the other identified genes in differentiating human ESCs. In total, these results show that an epigenetic and gene expression signature can be used to uncover the evolutionarily conserved genes which characterize the cardiac lineage in diverse species, and that the integration of orthologous data sets improves the predictive power of epigenetic findings alone.